A living body is protected from foreign substances mainly by an immune response, and an immune system has been established by various cells and the soluble factors produced thereby. Among them, leukocytes, especially lymphocytes, play a key role. The lymphocytes are classified in two major types, B lymphocyte (which may be hereinafter referred to as B cell) and T lymphocyte (which may be hereinafter referred to as T cell), both of which specifically recognize an antigen and act on the antigen to protect the living body.
T cell is subclassified to helper T cell having CD (Cluster of Differentiation)4 marker (which may be hereinafter referred to as TH), mainly involved in assisting in antibody production and induction of various immune responses, and cytotoxic T cell having CD8 marker (Tc: cytotoxic T lymphocyte, also referred to as killer T cell, which may be hereinafter referred to as CTL), mainly exhibiting a cytotoxic activity. CTL, which plays the most important role in recognizing, destroying and eliminating tumor cell, virus-infected cell or the like, does not produce an antibody specifically reacting with an antigen like B cell, but directly recognizes and acts on antigens (antigenic peptide) from a target cell which is associated with major histocompatibility complex [MHC, which may be also referred to as human leukocyte antigen (HLA) in human] Class I molecules existing on the surface of the target cell membrane. At this time, T cell receptor (hereinafter referred to as TCR) existing on the surface of the CTL membrane specifically recognizes the above-mentioned antigenic peptides and MHC Class I molecules, and determines whether the antigenic peptide is autologous or nonautologous. Target cell which has been determined to be nonautologous is then specifically destroyed and eliminated by CTL.
Recent years, a therapy which would cause a heavy physical burden on a patient, such as pharmacotherapy and radiotherapy, has been reconsidered, and an interest has increased in an immunotherapy with a light physical burden on a patient. Especially, there has been remarked an effectiveness of adoptive immunotherapy in which a lymphocyte such as CTL that specifically reacts with an antigen of interest is induced ex vivo from a lymphocyte derived from a human, or the above-mentioned lymphocyte is expanded without induction, and then transferred to a patient. For example, it has been suggested in an animal model that adoptive immunotherapy is an effective therapy for virus infection and tumor (for example, Non-Patent Publications 1 and 2). In this therapy, it is important to maintain or increase the number of the CTLs in a state in which the antigen-specific cytotoxic activity of the cells is maintained or enhanced.
In the adoptive immunotherapy as described above, it is necessary to administer cytotoxic lymphocytes in the number of cells of a given amount or larger in order to obtain a therapeutic effect. In other words, it can be said that it is the greatest problem to obtain the above number of cells ex vivo in a short period of time.
In order to maintain and enhance an antigen-specific cytotoxic activity of CTL, there has been generally employed a method of repeating stimulation with an antigen of interest when a specific response to an antigen for CTL is induced. However, in this method, the number of CTL finally obtained may usually be decreased, so that a sufficient number of cells cannot be obtained.
Next, regarding the preparation of the antigen-specific CTL, there has been each reported a method for isolating and expanding a CMV-specific CTL clone using autologous CMV infected fibroblast and IL-2 (for example, Non-Patent Publication 6) or using anti-CD3 monoclonal antibody (anti-CD3 mAb) and IL-2 (for example, Non-Patent Publication 7).
Furthermore, Patent Publication 1 discloses an REM method (rapid expansion method). This REM method is a method for expanding a primary T cell population containing antigen-specific CTLs and TH in a short period of time. In other words, this method is characterized in that a large amount of T cell can be provided by expanding individual T cell clones, and that the number of antigen-specific CTLs is increased using an anti-CD3 antibody, IL-2, and PBMCs (peripheral blood mononuclear cells) made deficient in an ability for expansion by irradiation, and Epstein-Barr virus (hereinafter abbreviated as EBV)-infected cells.
In addition, Patent Publication 2 discloses a modified REM method, wherein the method is a method using as feeder cells a nondividing mammal cell strain expressing a T-cell stimulating component which is distinguishable from PBMCs to reduce an amount of PBMCs used.
As lymphocytes which are effective for the treatment of a disease other than CTLs, there has been known, for example, lymphokine-activated cells (for example, Non-Patent Publication 3) and tumor-infiltrating lymphocytes (TILs) induced with interleukin-2 (IL-2) in a high concentration (for example, Non-Patent Publications 4 and 5).
The lymphokine-activated cells are a functional cell population having a cytotoxic activity, which are obtained by adding IL-2 to peripheral blood (peripheral blood leukocyte), umbilical cord blood, tissue fluid or the like containing lymphocytes, and culturing the cells in vitro for several days. In the step of culturing the lymphokine-activated cells, proliferation of the lymphokine-activated cells is further accelerated by adding an anti-CD3 antibody thereto. Further, it has been known that, in the culture step, a proliferation ratio of the cells is further improved by adding not only the anti-CD3 antibody but also an anti-CD28 antibody thereto to co-stimulate the cells (for example, Non-Patent Publication 8). The lymphokine-activated cells thus obtained have a cytotoxic activity non-specifically to various cancer cells and other targets.
Fibronectin is a gigantic glycoprotein having a molecular weight of 250 thousands, which exists in an animal blood, on the surface of a cultured cell, or in an extracellular matrix of a tissue, and has been known to have various functions. A domain structure thereof is divided into seven portions (FIG. 1 et seq), wherein three kinds of similar sequences are contained in an amino acid sequence thereof, repetitions of each of these sequences constituting the entire sequence. Three kinds of the similar sequences are referred to as type I, type II and type III. Among them, the type III is constituted by 71 to 96 amino acid residues, wherein an identity of these amino acid residues is 17 to 40%. In fibronectin, there are fourteen type III sequences, among which the 8th, 9th or 10th sequence (each being hereinafter referred to as III-8, III-9 or III-10) is contained in a cell binding domain, and the 12th, 13th or 14th sequence (each being hereinafter referred to as III-12, III-13 or III-14) is contained in a heparin binding domain. In addition, a VLA (very late activation antigen)-5 binding region is contained in III-10, and its core sequence is RGDS. In addition, a region referred to as IIICS exists at a C-terminal side of the heparin binding domain. A region referred to as CS-1 consisting of 25 amino acids and having a binding activity to VLA-4 exists in IIICS (for example, Non-Patent Publications 9 to 11).
In the preparation of the lymphokine-activated cells and the cytotoxic lymphocytes, an action of improving a proliferation ratio of the cells and an action of maintaining a cytotoxic activity by using fibronectin and a fragment thereof have been already studied by the present inventors (for example, Patent Publications 3, 4 and 5). However, considering the actual use for adoptive immunotherapy, the methods of the above-mentioned publications are not satisfactory at all, and a method for expanding lymphocytes with a further higher proliferation ratio of the cells has been desired.    Non-Patent Publication 1: authored by Greenberg, P. D., published in 1992, Advances in Immunology    Non-Patent Publication 2: Reusser P. and three others, Blood, 1991, 78(5), 1373-1380    Non-Patent Publication 3: Riddell S. A. and four others, J. Immunol., 1991, 146(8), 2795-2804    Non-Patent Publication 4: Riddell S. R. and one other, J. Immunol. Methods, 1990, 128(2), 189-201    Non-Patent Publication 5: Rosenberg S. A. et al., N. Engl. J. Med. 1987, 316(15), 889-897    Non-Patent Publication 6: Rosenberg S. A. et al., N. Engl. J. Med., 1988, 319(25), 1676-1680    Non-Patent Publication 7: Ho M. and nine others, Blood, 1993, 81(8), 2093-2101    Non-Patent Publication 8: Koberda J. and two others, J. Cancer Res. Clin. Oncol., 1993, 119(3), 131-136    Non-Patent Publication 9: authored by Deane F. Mosher, published in 1988, FIBRONECTIN, ACADEMIC PRESS INC., 1-24    Non-Patent Publication 10: Kimizuka F. and eight others, J. Biochem., 1991, 110(2), 284-291    Non-Patent Publication 11: Hancnberg H. and five others, Human Gene Therapy, 1997, 8(18), 2193-2206    Patent Publication 1: WO 96/06929    Patent Publication 2: WO 97/32970    Patent Publication 3: WO 03/016511    Patent Publication 4: WO 03/080817    Patent Publication 5: WO 2005/019450